ABSTRACT: Project 2 ? Targeted Fungal Ras Signaling for Antimicrobial Therapy Proteins of the important Ras signal transduction pathway have been intensely studied as targets for anticancer therapeutics, but also hold potential in other diseases. We have found central roles for Ras and Ras-processing enzymes in fungal pathogenesis, particularly related to pathogens' capability for high temperature growth. Our evidence suggests that inhibitors of the Ras pathway could serve as novel therapies to treat human fungal diseases. Numerous compounds are in preclinical development and in clinical trials that inhibit Ras, and we propose to start from these known inhibitors to design agents with improved antifungal properties based on our knowledge of the proteins' structures. Our targets are enzymes called prenyltransferases that modify Ras proteins, and we also will explore a second set of Ras-modifying enzymes, the palmitoyltransferases. Both of these proteins are required for proper localization of Ras signaling proteins; without the enzymes' activities, Ras function is lost. We have studied the structure of mammalian prenyltransferases and have helped develop focused prenyltransferase inhibitors. In more recent work, we also have solved the structures of four fungal prenyltransferases, FTase and GGTase, in different pathogenic fungi. Our work has revealed molecular features that are conserved in the fungal enzymes but differ from the mammalian ones. Such fungal-specific features suggest that current inhibitors could be derivatized for enhanced antifungal effect. Our data also indicate that Ras inhibition will be synergistic with therapies directed against fungal calcineurin and trehalose pathways (Projects 1 and 3). In this proposal, we build on and expand our structure-based studies to develop novel synergistic, broad- spectrum antifungals. We propose three Specific Aims: 1) To redirect the large collections of FTase inhibitors (FTIs) developed for cancer chemotherapeutics to antifungal agents using structure-guided approaches. 2) To investigate GGTase as an antifungal drug target, and similarly develop potent inhibitors using structure-guided approaches. 3) To investigate palmitoylation of Ras-family proteins in fungal pathogenesis, and evaluate its potential for therapeutic targeting. Palmitoyltransferases are highly divergent between fungi and mammals, and our proposed genetic and biochemical studies will allow the focused screening and optimization of palmitoylation inhibitors as new antifungals. This Project will utilize all three Cores and interact closely with Projects 1 and 2 of this Program Project in order to accelerate development of novel agents to treat life- threatening fungal infections in humans.